Goat blood samples were obtained from #FG4592 randurls[1|1|,|CHEM1|]# Chama district in Zambia. The former two sites are endemic for East Coast fever caused by Theileria parva, and the latter are endemic for trypanosomiosis. These areas are habitats for Amblyomma ticks and lacked adequate tick control programs. In total, 150 bovine blood samples, 50 from each site, and 35 goat blood samples were used in the present study. In addition, this study employed DNA samples extracted from the blood of lambs at Kerr Seringe in the Gambia, where heartwater is endemic. Nineteen samples were
randomly selected from those used in the previous study, some of which were positive by pCS20 nested PCR [17]. As positive controls, four blood samples obtained from two sheep experimentally infected with E. ruminantium Senegal isolate were used. Blood was collected from each sheep on days 14 and 16 post infections when the animals showed high fever. Research on samples from animals was conducted adhering to guidelines
for Care and Use of Laboratory Animals and was approved by the Animal Care and Use Committee of the Utrecht University. DNA extraction DNAs from rickettsia-infected cell cultures were extracted using Nucleospin Tissue kits (Macherey-Nagel, Duren, Germany). A. variegatum ticks EPZ004777 mouse were washed with 70% ethanol and rinsed twice with distilled water. Tick samples were then homogenized by Micro Smash MS-100R (TOMY, Tokyo, Japan) for 2 min at 2,500 rpm, followed by DNA extraction with DNAzol (Invitrogen, Carlsbad, CA). DNAs from blood were extracted using either the GenTLE kit (Takara, Shiga, Japan) or a DNA isolation kit for mammalian blood (Roche, Mannheim, Germany). All procedures were carried out as described by the manufacturers. LAMP primers Two sets of LAMP primers were designed for the pCS20 and sodB genes
of E. ruminantium. The nucleotide sequence of the Welgevonden isolate of E. ruminantium was retrieved from GenBank [GenBank:CR767821] and aligned with the available sequences of other isolates to identify Endonuclease conserved regions, using CLUSTALW software version 1.83 (DNA Data Bank of Japan; http://clustalw.ddbj.nig.ac.jp/top-e.html). A potential target region was selected from the aligned sequences, and four primers, comprising two outer (F3 and B3) and two inner (FIP and BIP) primers, were designed using LAMP primer software PrimerExplorer V4 (http://primerexplorer.jp/elamp4.0.0/index.html; Eiken Chemical Co., Japan). Loop primers (LF and LB) were designed manually. The designed primer sequences are shown in Table 5.